Pseudomonas aeruginosa is a common and extremely virulent cause of serious infections in immune-compromised/suppressed patients (e.g., HIV and cancer), cystic fibrosis patients, and those on mechanical ventilation or with burn wounds. Frequent antibiotic resistance and the highly virulent nature of P. aeruginosa make it deadlier than most other bacteria. New chemical classes of antibiotics acting on novel accessible targets are crucial for continued effective therapy against P. aeruginosa because such drugs will not be subject to existing resistance mechanisms. The strategy of this project is to develop new drugs by screening a diverse collection of synthetic and natural product compounds against extra-cellular targets that are critical for virulence. The type III secretion system (TTSS), dedicated to the secretion of protein toxins ("effectors") and their translocation into the cytoplasm of human cells has been validated as a clinically important target in P. aeruginosa. Drugs targeting TTSS may be used in combination with bactericidal antibiotics in immune-compromised patients, and they may be useful on their own in patients with competent immune systems or for prophylactic application. The goal of this project is to identify specific inhibitors of TTSS and to develop them into novel antibiotics for therapy against P. aeruginosa. In Phase I, 2 new whole-cell reporter assays for the TTSS of P. aeruginosa will be developed. The primary assay for high throughput screening will detect inhibitors of secretion and translocation of an effector-B-lactamase chimeric reporter via TTSS into CHO cells. The second assay will be based on the tight linkage between TTSS gene expression and the activity of the secretion channel by building a transcriptional luciferase reporter which is down-regulated by 100-fold upon loss of TTSS function. A diverse chemical library will be screened with the primary assay, hits validated by the transcriptional reporter assay as well as by a TTSS-dependent cytotoxicity assay. Validated hits will be prioritized by demonstrating minimal inhibition of both bacterial cell growth and mammalian cell viability, minimal disruption of cell membranes, and by determining the spectrum of TTSS inhibition. We will accomplish the following specific aims: (1) Develop a high-throughput secreted reporter screen for inhibitors of P. aeruginosa TTSS; (2) Develop a transcriptional reporter secondary assay for inhibitors of P. aeruginosa TTSS; (3) Screen a diverse compound library to identify and validate TTSS inhibitors; and (4) Prioritize validated screening hits for in vitro potency, mechanism, spectrum, and selectivity. [unreadable] [unreadable]